Abstract This K99/R00 grant will facilitate the applicant's (Dr. Nagaraj Kerur) long-term goal to develop as an independent investigator in vision research. Geographic atrophy (GA) is an advanced form of age-related macular degeneration (AMD) characterized by central loss of vision due to retinal pigmented epithelium (RPE) degeneration. Currently there is no therapy available for GA because of its unclear etiology. We recently discovered that DICER1 deficit causes a pathologic accumulation of Alu RNA transcripts in the RPE of human GA eyes and that the accumulated Alu RNAs activate the NLRP3 inflammasome, which in turn leads to IL- 18/MyD88-dependent RPE cell death (Kaneko et al., Nature 2011; Tarallo et al., Cell 2012). In exciting, new preliminary studies we have discovered that Alu RNA-induced NLRP3 inflammasome activation occurs in a non-canonical fashion- a newly described pathway in which caspase 4 (aka caspase 11 in mouse) is required for the NLRP3 inflammasome activation (in contrast the canonical inflammasome activation is independent of caspase 4). The central goal of the proposed research is to define role of caspase 4-mediated non-canonical NLRP3 inflammasome activation in the context of GA and to identify critical signaling pathways regulating caspase 4 activation via the following aims: (1) Define the signaling pathways that regulate caspase 4 (aka caspase 11 in mouse) activation by Alu RNA; (2) Elucidate the mechanisms of caspase 4 (aka caspase 11 in mouse) activation of the NLRP3 inflammasome; and (3) Measure the activation of these non-canonical inflammasome signaling pathways in human GA. These studies will illuminate the molecular foundation of GA, unravel novel regulatory checkpoints of NLRP3 inflammasome activation, and provide a strong molecular foundation for exploiting new therapeutic targets in AMD. During the mentored phase, (K99) the applicant will carry out the proposed studies under direct supervision of the mentor Dr. Jayakrishna Ambati at the University of Kentucky. In addition, an advisory committee consisting of co-mentor, Dr. Katherine Fitzgerald and the faculty members at the Department of Ophthalmology and Visual Sciences will monitor the applicant's research progress and offer assistance in advancing the applicant's transition towards independent investigator. The Ambati Group at the University of Kentucky as well as the Fitzgerald Laboratory at the University of Massachusetts Medical School, both offer scientifically enriching environments and resources necessary for the advancement of the applicant's scientific career. The major focus of K99 phase will include: (1) mentored research to define molecular insights into the caspase 4 activation mechanism and to optimize experimental conditions for studies to be carried out in the R00 phase; and (2) training activities consisting of formal course work, seminars, workshops on grant writing and acquisition of other skill-sets required for successfully developing and leading an independent research program. During the R00 phase, the focus will be on: (1) deciphering the mechanisms by which caspase 4 regulates NLRP3 inflammasome activation; and (2) quantifying the activation of non-canonical inflammasome signaling pathways in human GA eyes. Overall this proposal will not only unravel the novel molecular mechanisms of RPE degeneration in GA, but will also equip the applicant with suitable skills to transition into an independent career in vision research.